TABLE 1.
Kinetic parameters of T. brucei TK
Km and Vmax values with S.D. are calculated from fitting the Michaelis-Menten curve to a hyperbola by nonlinear regression using the GraphPad Prism software.
| Km | Vmax | kcat | kcat/Km | |
|---|---|---|---|---|
| μm | μmol × min−1 × mg−1 | s−1 | s−1 × m−1 | |
| Full-length TK | ||||
| ATP, dThd (0.5 μm)a | 180 ± 30 | 0.190 ± 0.009 | ||
| ATP, dThd (5 μm)a | 220 ± 20 | 1.9 ± 0.05 | ||
| ATP, dThd (50 μm)a | 210 ± 30 | 3.6 ± 0.10 | 3.1 | 1.5 × 104 |
| ATP, dIno (0.5 mm)a | 320 ± 60 | 0.095 ± 0.007 | ||
| dThd | 7.8 ± 0.9 | 3.6 ± 0.10 | 3.1 | 4.0 × 105 |
| 5-Fluoro-dUrd | 30 ± 4 | 4.2 ± 0.10 | 3.6 | 1.2 × 105 |
| dUrd | 80 ± 5 | 5.9 ± 0.09 | 5.1 | 6.3 × 104 |
| dCyd | 0.029b | |||
| Urd | 7800 ± 900 | 1.7 ± 0.07 | 1.5 | 190 |
| dIno | 2500 ± 300 | 0.93 ± 0.07 | 0.80 | 320 |
| dGuo | 2600 ± 200 | 0.39 ± 0.010 | 0.34 | 130 |
| dAdo | 8400 ± 700 | 0.055 ± 0.003 | 0.047 | 5.6 |
| TK Domain 2 | ||||
| ATP, dThd (50 μm)a | 98 ± 15 | 8.2 ± 0.3 | 3.9 | 4.0 × 104 |
| dThd | 46 ± 2 | 7.3 ± 0.10 | 3.5 | 7.6 × 104 |
| dUrd | 430 ± 40 | 11.2 ± 0.3 | 5.3 | 1.2 × 104 |
a Experiments to calculate kinetic constants for ATP were performed using thymidine (at a concentration of 0.5, 5 or 50 μm) or 0.5 mm deoxyinosine as a deoxynucleoside substrate. When ATP is the studied substrate, kcat and kcat/Km values are only shown in the cases where the deoxynucleoside concentration is high enough to saturate the enzyme activity.
b The value represents the enzyme activity with 10 mm substrate concentration.